For the past 10 years, the Principal Investigator's laboratory has been interested in the hydroxylation of medium-chain fatty acids and prostaglandins by kidney, liver, and lung microsomal preparations and purified preparations of cytochromes P-450 derived from these tissues. Hydroxylation of lauric acid and prostaglandins E1, A1, and F2Alpha occurs mainly in the Omega- and (Omega-1)-positions leading to the production of products which can be further oxidized by cytosolic dehydrogenases to dicarboxylic acids. The formation of high concentrations of Omega-hydroxylated products of prostaglandins E1 and F2Alpha by lung microsomes from pregnant rabbits and the occurrence of (Omega-1)-hydroxyprostaglandin E1 in primate semen at high levels has led to increased interest in the metabolism of these prostaglandins in reproductive biology. Experiments are being proposed to: 1) localize the cytochrome(s) P-450 in lung tissue responsible for the Omega- hydroxylase activities by fluorescent antibody labelling and/or colloidal gold-labelled antibody techniques; 2) determine the fate of the Omega-hydroxylated metabolites of fatty acids and prostaglandins by lung perfusion techniques; 3) test the physiological activities of the Omega-hydroxyprostaglandins vs the parent compounds; 4) determine the specificity of the Omega-hydroxylase(s) in purified, reconstituted systems for prostaglandins and their precursors in the presence and absence of cytochrome b5; 5) localize the male reproductive organ responsible for the formation of and determine the precursor(s) of the (Omega-1)-hydroxy PGE1 in male primate semen; 6) isolate and purify the (Omega-1)-hydroxylation system from the appropriate organ if localization is possible and tissue is obtainable; 7) utilization of the antibody to lung microsomal prostaglandin Omega-hydroxylase cytochrome P-450 (P-450 PG-Omega) to isolate the in vitro translation products of total RNA isolated from pregnancy- or progesterone-induced rabbits and isolation of specific mRNAs to obtain a cDNA probe for partial sequence analysis and comparison to other cytochromes P-450; and 8) cloning of the specific cDNA sequence of cytochrome P-450PG-Omega selected by the hybrid arrest technique and confirmed by the positive hybridization-translation assay. These studies are designed to determine the metabolism of, mechanism of action of, and molecular regulation for Omega- and (Omega-1)-hydroxylated prostaglandins E1 and F2Alpha.